The present invention relates to the provision of a biologically safe hemolymph sera, preferably hemocyanin, more preferably KLH (keyhole limpet hemocyanin).
Hemocyanin is a blue copper protein which occurs in a freely dissolved form in the blood of numerous molluscs and arthropods and transports oxygen. Of the molluscs, the cephalopods, chitons, most gastropods and some bivalves contain hemocyanin. Among the arthropods, hemocyanin is typical of arachnids, xiphosurans, malacostracan crustaceans and Scutigera. Numerous species of insects contain proteins which are derived from hemocyanin. Hemocyanins are present in the extracellular medium and float in the hemolymph.
While arthropod hemocyanin has a maximum diameter of 25 nm under an electron microscope and a subunit has a molecular weight of 75,000 Dalton (Da), mollusc cyanins are much larger. Thus e.g. the hemocyanin of Megathura has a diameter of 35 nm and is composed of 2 subunits. Each subunit has a molecular weight of approx. 400,000 Da and is divided into eight oxygen-binding domains, each of which has a molecular weight of approx. 50,000. The domains differ immunologically.
The hemocyanin of gastropods visible under an electron microscope has a molecular weight of approx. 8 million Da and is a didecamer. In contrast to this, the hemocyanin of cephalopods is arranged as an isolated decamer, which also differs significantly from the hemocyanin of gastropods in the quaternary structure.
Traditionally, hemocyanin was obtained from hemolymph from the Megathura crenulata. More recently, the market for gastropod hemocyanins has expanded to include hemocyanin from Haliotis tuberculata and Concholepus concholepus. The hemolymph from other gastropod molluscs is also under investigation for useful properties.
The hemocyanin of the Californian keyhole limpet Megathura crenulata is of particular immunological interest. The hemocyanin is therefore also called keyhole limpet hemocyanin (KLH). Hemocyanins are very potent antigens. Immunization of a vertebrate leads to a non-specific activation of the immune system which to date is not very well understood. By the general activation of the immune system, it is then possible also to achieve an immune reaction to other foreign structures which have previously been tolerated. KLH is used above all as a hapten carrier in order thus to achieve the formation of antibodies against the hapten.
In addition to Megathura crenulata, the abalone Haliotis tuberculata also belongs to the Archaegastropoda group, which is relatively old in respect of evolution. It is known that Haliotis also produces hemocyanin.
Native KLH is found in the hemolymph (pH 6.0-8.0) in colloidal solution as a didecamer (molecular weight: around 8 million Da) and as multidecamers (molecular weight: 12 to about 32 million Da). The quantitative distribution of these aggregates varies. The didecamers and multidecamers of KLH are composed of 2 types of subunits with an average molecular weight of around 400,000 Da. The two different types of subunits as well as the two different aggregation types are due to the fact that native KLH is a mixture of two different types KLH 1 and KLH 2.
KLH is a mixture of two different hemocyanins, which are called KLH1 and KLH2. The subunit of KLH1 is a 390 kDa polypeptide which consists of eight globular domains called 1 a to 1 h according to their sequence in the subunit. On the other hand, KLH2 has a molecular weight of 360 kDa and according to the most recent data also contains 8 domains, called 2 a to 2 h. In vivo every type of subunit forms homo-oligomers, while no hetero-oligomers have been observed.
Hemocyanins may be obtained in farms from test animals. Methods described for collection of hemolymph involve inserting a needle into a muscle of the foot to penetrate the pedal blood sinus (Harris et al., “Keyhole Limpet Haemocyanin: Negative Staining in the Presence of Trehalose,” Micron, 26 (1): 25-33 (1995). Semi-automated systems were established allowing the collection of high amounts of hemolymph without killing the animals. The manufacturing procedures allow extracting commercial quantities of hemocyanin from animals grown in a controlled environment (WO 02/085389, US2002/192633).
There are a variety of well-known methods for purifying hemocyanins from crude hemolymph, which is the biological source of hemocyanins. These methods include differential centrifugation, gel-permeation chromatography, and ion-exchange chromatography (U.S. Pat. No. 5,407,912). Purified hemocyanins are commercially available in many forms.
The incorporation of hemocyanins into promising new therapeutic products (see e.g., Jurincic-Winkler et al., “Antibody Response to Keyhole Limpet Hemocyanin (KLH) Treatment in Patients with Superficial Bladder Carcinoma,” Anticancer Res., 16 (4A): 2105-10 (1996); and Biomira, Inc. Company Press Release, Biomira.com, 2001) has resulted in the need for a sustainable supply of commercial quantities of hemocyanin produced under conditions that meet the health and safety standards imposed by the United States Food and Drug Administration and other regulatory agencies.